| Oil content in seeds is definitely the critical objective for rapeseed breeding. However, accumulation of seed oil is a complicated metabolic and genetic regulatory process during seed development. It has been well documented in Arabidopsis that hundreds of genes were involved in Acyl Lipid Metabolism pathways, which makes difficulty to understand its genetic inheritance. Up to date, a number of QTLs for oil content in rapeseed have been identified, but fine mapping and map based cloning for them have not yet been reported. The current study were based on the previous mapping analysis of oil content in multiply environments and aimed at fine mapping a most significant QTL for oil content in linkage group 7 and searched for candidate gene/genes underlying this QTL.Brassica rapa BAC sequences were selected for locus specifical marker design. Near-isogenic lines (NILs) containing introgressions from donor parent "Gaoyou" were developed using advanced backcross QTL approach to get BC3F1/BC3F2, BC3F2/BC3F3 and BC4F1/BC4F2 progenies. The single plants of BC3F1, BC3F2 and BC4F1 populations were genotyped in each of marker loci within the target region, while phenotypic data were obtained from respective selfing progenies BC3F2, BC3F3and BC4F2 lines. They were tested in field with two replications and seed oil content was determined by near-infrared reflectance spectroscopy (NIRS) for three times per line.After initial QTL mapping for oil content in multiply environments with SG-DH population (282 lines), we selected the most significant one in A7 (OilA7) as the target for fine mapping and map-based cloning. OilA7 was detected significantly in all eleven locations with the LOD value from 3.09 to 20.54, which could explain 3.59% to 24.69% of the total phenotypic variation. We first determined the genotypes of 2500 BC3F1 plants by 11 markers covering the whole QTL region of 21.1cM interval. The QTL validation was performed using corresponding 1147 BC3F2 lines and OilA7 was mapped in a 12.6 cM region between the markers ZAAS176 and EM4ME8. The further fine mapping was carried out applying 1300 BC3F2/ BC3F3 and 1500 BC4F,/BC4F2 families and newly developed 24 markers between ZAAS176 and EM4ME8. OilA7 was then localized to a high-resolution linkage map by progeny testing of homozygous recombinant lines and narrowed the locus to about 7.3cM (BC3F2/BC3F3) and 528kb (BC4F1/ BC4F2) regions, respectively. The comparative analysis of oil content between homozygous BC4F2 sister sub-NILs carrying "Gaoyou" fragment (n=16,46.4%) and NILs containing "Sollux" segment (n=17,44.3%) in the 528kb target region showed significant difference of 2.1%(p=0.000) in oil content.Arabidopsis (E< 10"20), which could be grouped into one hundred and eight gene families. One highly homologous (E=1.3×10-57) lipid synthesis related gene was found lying in the region and might be considered as candidate gene for functional analysis.Present study validated the major QTL for oil content in linkage group 7 and further fine mapped it in a 528kb genomic region. Our results indicated that the homozygous substitution of European cultivar Sollux alleles by Chinese material Gaoyou's alleles in OilA7 might increase oil content in seeds for about 2.0%, indicating OilA7 is an important QTL controlling oil content in rapeseed and cloning for gene/genes underlying the QTL is of particular interest for breeding program. |
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